It is generally recognized that there are two infectious forms of baculoviruses. Early in the replication cycle, baculoviruses produce nucleocapsids in the nucleus of a host cell which bud through the nuclear membrane into the cytoplasm and subsequently bud through the plasma membrane. These extracellular virus particles are referred to as budded virus or nonoccluded virus (NOV). The NOV are highly infectious to susceptible insect tissue culture cells. Insect larvae can be infected by injection of NOV into the hemocoel (circulatory system). However, NOV is generally recognized as non-infectious when administered per os (feeding) to larvae. Generally, only very low infections rates can be achieved with high concentrations of NOV using per os larval inoculations. Accordingly, the NOV have been used to infect larvae per os only in rare instances and in the absence of other alternatives.
Late in a baculovirus replication cycle, the nucleocapsids do not bud through the nuclear membrane, and they become membrane bound within the cell nucleus. At the same time baculovirus polyhedrin or granulin genes are transcribed and translated producing large amounts of protein which crystallize around the membrane-bound nucleocapsids present in the nucleus. These protein crystals containing virus particles have historically been referred to as granules with granulosis viruses and polyhedra with nuclear polyhedrosis viruses (both are types of baculoviruses). It is well known in the art that granulin and polyhedrin are functionally identical and are maintained in baculoviruses that are functional identical and have been termed differently due to historical precedent.
The granules and polyhedra of baculoviruses are used to efficiently infect susceptible host larvae through per os inoculations. The alkaline pH in the larval gut region results in dissolution of the crystalline protein surrounding the occluded virus (OV) particles. Following release from the crystal, the OV particles are able to efficiently infect larval gut cells. Although highly infectious per os, the OV particles are generally recognized as having an extremely low potential to initiate infections with tissue culture cells or by injection into the hemocoel of susceptible host larvae.
Accordingly, the baculovirus literature reflects the use of NOV as the form of inoculum to infect host larvae by hemocoelic injections or to infect insect tissue culture cells by addition to cell culture medium. And the polyhedra containing OV are used to infect susceptible host larvae by per os inoculations.
The construction of most baculovirus expression vector systems (BEV) has been based on replacement of the polyhedrin gene coding region with a foreign gene under the transcriptional control of the polyhedrin gene promoter (Pennock, G. D., Shoemaker, C., Miller, L. K. 1984, "Strong and regulated expression of Escherichia coli b-galactosidase in insect cells with a baculovirus vector", Mol. and Cell. Bio. 4:399-406; and Smith, G. E., Summers, M. D. and Fraser, M. J. 1983, "Production of human beta interferon in insect cells infected with a baculovirus expression vector", Molecular and Cell. Biol. 3:2156-2165). To date, hundreds of foreign genes have been expressed in BEV [Luckow, V. A. 1990, "Cloning and expression of heterologous genes in insect Cells with baculovirus vectors in Recombinant DNA Technology and Applications", (C. Ho, A. Prokop and R. Bajpai, eds.) McGraw-Hill, New York]. While the goal of most researchers has been to produce a protein for further study research or as a commercial product, there has been an increased interest in exploiting the BEV to express foreign proteins which would improve the pesticidal properties of the recombinant baculoviruses (Carbonell, L. F., Hodge, M. R., Tomalski, M. D., Miller, M. K., 1988, "Synthesis of a gene coding for an insect-specific scorpion neurotoxin and attempts to express it using baculovirus vectors", Gene 73:409-418; Hammock, B. D., Bonning, B. C., Possee, R. D., Hanzlik, T. N., Maeda, S., 1990, "Expression and effects of the juvenile hormone esterase in a baculovirus vector", Nature 344:458-461; Maeda, S., 1989, "Increased insecticidal effect by a recombinant baculovirus carrying a synthetic hormone gene", Biochem. Biophys. Res. Comm. 165: 1177-1183; Maeda, S., Volrath, S. L., Hanzlik, T. N., Harper, S. A., Majima, K., Maddox, D. W., Hammock, B. D. and Fowler, E., 1991, "Insecticidal effects of an insect-specific neurotoxin expressed by a recombinant baculovirus", Virol. 184:777-780; Merryweather, A. T., Weyer, J., Harris, M. P. G., Hirst, M., Booth, T., Possee, R. D., 1990, "Construction of genetically engineered baculovirus insecticides containing the Bacillus thuringiensis ssp. kurstaki HD-73 delta endotoxin", J. Gen. Virol. 71:1534-1544; O'Reilly, D. R., Miller, L. K., 1989, "A Baculovirus blocks insect molting by producing ecdysteroid UDP-glucosyl transferase", Science 245:1110-1112; Stewart, L. M. D., Hirst, M., Ferber, M. L., Merryweather, A. T., Cayley, P. J., and Possee, R. D., 1991, "Construction of an improved baculovirus insecticide containing an insect-specific toxin gene", Nature 352:85-88; Tomalski, M. D. and Miller, L. K., 1991, "Insect paralysis by baculovirus-mediated expression of a mite neurotoxin gene", Nature 352:82-85; and O'Reilly, D. R. and Miller, L. K., 1991, "Improvement of a baculovirus pesticide by deletion of the EGT gene", Biotechnology 9:1086-1089). Baculoviruses are natural pathogens of many agriculturally important insect pests and are among the most promising alternatives to synthetic chemical pesticides (Wood, H. A. and R. R. Granados, 1991, "Genetically engineered baculoviruses as agents for pest control", Ann. Rev. Microbiol. 45:69-87).
In order to assess the pesticidal properties of BEV, insect host larvae must be inoculated at known virus dosages during a specified period of time and the time course of infection followed. Deletion of the polyhedrin gene from BEV and the resultant lack of polyhedra production (and, therefore, the resultant absence of OVs) has been problematic to performing larval bioassays because the naturally occurring NOV is not very infections per os. Additionally, it has been proposed to infect insect larvae with BEV to produce proteins of commercial interest (Maeda, S., Kawai, T., Obinata, M., Fujiwara, H., Horiuchi, T., Seki, Y., Sato, Y., and Furusawa, M., 1985, "Production of human alpha-interferon in silkworm using a baculovirus vector", Nature 315:592-594). Currently as demonstrated in all the references listed above, the common method of infecting larvae with BEV containing pesticidal or other foreign genes but lacking a functional polyhedrin gene (polyhedrin-minus virus) is by hemocoelic injection of larvae with the NOV. Alternatively, diet contamination with NOV has been used to establish infections, but very large doses are required due to the poor infectivity of this form of the virus by the oral route [Granados, R. R. and Williams, K. A., 1986, "In vivo infection and replication of baculoviruses" in The Biology of Baculoviruses Vol. I, "Biological Properties and Molecular Biology" (R. R. Granados and B. A. Federici, eds.), CRC Press, Boca Raton, Fla.].
The emphasis in the art has been on meeting the need for a method to simply and efficiently infect host insect larvae with polyhedrin-minus BEV isolates using per os inoculation procedures. The development of such a method would provide an efficient and simple means to: a) evaluate the pesticidal properties of a polyhedrin-minus BEV expressing foreign pesticidal gene products, b) infect large numbers of host insect larvae with polyhedrin-minus BEV expressing foreign proteins of commercial value (thereby, providing an alternative to BEV protein production in insect tissue culture cells), and c) to use polyhedrin-minus BEV expressing foreign pesticidal gene products as pesticides under field conditions.
The production of commercial protein products in insect larvae following infection with a polyhedrin-minus BEV currently would require larval NOV injections, a costly and laborious process. The commercial pesticide application of a polyhedrin-minus BEV expressing pesticidal proteins has been considered not possible by those schooled in the art. Such a pesticidal product would be ideal in that the progeny NOV (no polyhedra formed) would not persist in the environment [Bishop, D. H. L., Entwistle, P. F., Cameron, I. R., Allen, C. J., Possee, R. D., 1988, "Field trials of genetically-engineered baculovirus insecticides", in The Release of Genetically-engineered Micro-organisms, (eds. M. Sussman, C. H. Collins, F. A. Skinner, D. E. Stewart-Tull), Academic Press, N.Y.; Hamblin, M., van Beek, N. A. M., Hughes, P. R., and Wood, H. A., 1990 "Co-occlusion and persistence of a baculovirus mutant lacking the polyhedrin gene", Applied and Environ. Microbiol. 56:3057-3062]. Because of the teachings of the current art, skilled artisans have directed much of their energies at either co-occlusion of polyhedrin-minus BEV with wild type virions (see above Hamblin et al. 1990, Miller, U.S. Pat. No. 5,071,748, Issued 12-10-91, Filed May 15, 1989, and ACS and Price, International Application Published Under The Patent Cooperation Treaty, International Publication Number WO 90/01556, International Publication Date February 22, 1990, International Application Number PCT/US89/03542) or constructing BEV which express foreign genes and a polyhedrin gene (polyhedrin-plus). The use of a polyhedrin-plus BEV as a commercial pesticide raises serious questions regarding persistence and competition of an engineered virus in the environment, from both ecological and health-safety standpoints.
It has been known in the art that late in the replication cycle of occluded baculoviruses, nucleocapsids become enveloped in bundles within the nucleus prior to being occluded by polyhedrin or granulin protein [Federici, B. A. 1986, Ultrastructure of baculoviruses in "The Biology of Baculoviruses Vol. I, Biological Properties and Molecular Biology" (R. R. Granados and B. A. Federici, eds.), CRC Press, Boca Raton, Fla.]. These virions are equally infectious per os when fed in polyhedra or following alkaline release and purification from the polyhedrin protein (Van Beck, N. A. M., Derksen, A. C. G., Granados, R. R. and Hughes, P. R., 1987, "Alkaline liberated baculoviruses particles retained their infectivity per os for neonate lepidopterous larvae", J. Invertebr. Pathol. 50:339-340). However, prior to this invention it was not known that these membrane bound virus particles destined to be occluded [which we refer to as pre-occluded virus (POV) particles], but not occluded if it derives from a polyhedrin-minus virus, were infectious per os. Furthermore, it was assumed in the art that infectious POV did not develop during replication of a polyhedrin-minus baculovirus; the continual and exhausting efforts in the art directed towards constructing pesticidal BEV with a polyhedrin gene in order to evaluate the biological properties as well as performing infectivity assays of polyhedfin-minus BEV by NOV injections or diet surface contamination with high concentrations of NOV illustrates the assumption of the non-existence of an infectious POV during replication of a polyhedfin-minus baculovirus.
In the publication entitled Co-Occlusion and Persistence of a Baculovirus Mutant Lacking the Polyhedrin Gene (Applied and Environmental Microbiology, Vol. 56, No. 10, October 1990, p. 3057-3062), Hamblin et al theorized that the secondary inoculum contained two types of virus particles: ExtraCellular Virus (ECV) in the hemolyph and intracellular virions which would normally be occluded in polyhedra. They believed they existed because they made the assumption that the infectivity shown may not have been possible from the ECV form of the virus. However, no experiments were performed to determine whether the observed infectivity was a result of the ECV, the intracellular virions or some other explanation. Even though some infectivity was shown, the inoculum was unstable and lost infectivity very rapidly. They came to the conclusion that in the absence of formation of polyhedra, these intracellular virions would not serve as an efficient inoculum. This conclusion is positively stated before and after the discussion of the theorized intracellular virions such that the paper as a whole teaches non-occluded virus cannot serve efficiently as a pesticide inoculum in the field. Hamblin et al did not teach how to isolate a POV particle, how to use a POV particle or how to differentiate a POV particle from any other form of a virus. Most importantly, Hamblin et al did not teach how to stabilize a POV particle so that it was infectious for more than a day after harvest.
Stabilizing virus particles can be quite difficult. Stabilization techniques that work for one virus may be disasterous for another virus. While freezing or lyopholization (freeze drying) have been used successfully to stabilize virus preparations, these techniques often fail or significantly reduce infectivity. Most studies that report stabilization by lyopholization do not report any data on the loss of infectivity. For instance, it is only important with vaccine preparations to retain some infectivity, but a loss of relative infectivity is not important. However, for large scale production of a pesticide, any loss in infectivity is crucial because that much more product needs to be prepared to accomplish the same task.
LeBlanc & Overstreet reported that desiccation for 48 hours inactivated Baculovirus penaei. (see "Effect of Desiccation, pH, Heat & Ultraviolet Irradiation on Viability of Baculovirus penaei", Journal of Invertebrate Pathology 57, 277-286 (1991)) David et al. showed that dry deposits of the granulosis virus of Pieris brassicae lost a significant amount of activity in only 2 days at 20.degree. C. (see "The Stability of a Purified Granulosis Virus of the European Cabbageworm, Pieris brassicae, in Dry Deposits of Intact Capsules", Journal of Invertebrate Pathology, 17, 228-233 (1971))
In some respects, POV particles are similar to non-occluded baculoviruses. Momoyamka showed that Baculoviral mid-gut Gland necrosis BMN virus (nonoccluded baculovirus) was inactivated within 1.5 hours after drying at about 30.degree. C. (see "Inactivation of Baculoviral mid-gut Gland necrosis BMN virus by UV Irradiation, Sunlight Exposure, Heating & Drying", Fish Pathol. 24(2), 115-118,(1989))